1. Field of the Invention
The present invention relates generally to the field of immunology and, more particularly, to processes for increasing the antigen binding capacity of class I and class II MHC molecules and to compositions of MHC molecules that are substantially free of such antigens.
2. Brief Description of the Relevant Art
Major histocompatibility complex (MHC) molecules (glycoproteins) are expressed on cells of higher vertebrates and play a role in immune responses. These molecules were discovered as a result of tissue grafting experiments wherein it was shown that graft rejection is an immune response to foreign antigens on the surface of the grafted cells. In humans, MHC molecules are referred to as HLA (human-leukocyte-associated) antigens because they were first identified in leukocytes. In mice, they are designated H-2 antigens.
MHC molecules are divided into two groups, class I and class II, which differ structurally and functionally from each other. In general, the major function of MHC molecules is to bind antigenic peptides and to display them on the surface of cells. These peptides result from an antigen presenting cell (APC) processing an antigen into peptide fragments, which can be as short as 10 to 20 amino acids. Class I MHC molecules are expressed on almost all nucleated cells and are recognized by cytotoxic T lymphocytes, which then destroy the antigen-bearing cells. Class II MHC molecules are expressed primarily on cells involved in immune responses, such as T lymphocytes, B lymphocytes, macrophages, etc. Class II MHC molecules are recognized by helper T lymphocytes and induce proliferation of helper T lymphocytes and amplification of the immune response to the particular antigenic peptide that is displayed.
It is thought that an antigenic peptide first forms a complex with either a class I or class II MHC molecule, and this complex fits into a single recognition site in either a cytotoxic or helper T lymphocyte receptor, respectively. In addition to binding antigenic peptides, MHC molecules also can bind with autologous, or "self" peptides. If T lymphocytes then respond to cells presenting "self" peptides, a condition of autoimmunity results. For a general discussion of the function of MHC molecules, see Grey, H. M., et al., Scientific American November, 1989:56-64 (incorporated herein by reference).
MHC molecule-antigenic peptide complexes are extremely stable. It has been found that the rates of peptide binding (on-rate or association) and unbinding (off-rate or disassociation) are extremely slow, see Buus, S., et al., Proc. Natl. Acad. Sci. USA 83:3968-3971 (1986); Sadegh-Nasseri, S., et al Nature 338:274-276 (1989) (these publications are incorporated herein by reference), and that only a small fraction of isolated MHC molecules are able to bind to added peptides, even when such peptides are present in high concentration and for a long incubation time, see Watts, T. H., et al , Proc. Natl. Acad. Sci. USA 83:9660-9664 (1986); Buus, S., et 21, Science 235:1353-1358 (1987); Adorini, L., et al., Nature 334:623-625 (1988) (these publications are incorporated herein by reference). This may be due to the fact that many of the MHC molecules still have peptides bound to them, even after the molecules have been isolated, see Demotz, S., et al., Nature 342:682-684 (1989); Wallny, H. J., et al., Nature 343:275-278 (1990) (these publications are incorporated herein by reference)
Difficulties in removing peptides bound to MHC molecules have stymied research on, for example, possible drug candidates to combat autoimmune conditions in the case where MHC molecules are bound to autologous proteins and also on screening methods for detecting certain antigenic peptides bound to the molecules. Although peptides have been removed from class II MHC molecules through inducing a change in pH, see Demotz, S., et al., Nature 342:682-364 (1989) (incorporated herein by reference), research has been centered on the peptides themselves and not the MHC molecules.
The unfolding and refolding of proteins, other than MHC glycoproteins, has been reported, see Jaenicke, R. Prog. Biophys. molec. Biol. 49:117-237 (1987) (incorporated herein by reference) for a review of such work, and the thermodynamic and kinetic mechanisms have been studied and folding intermediates have been characterized, see Harrison S. C., et al., Proc. Natl Acad Sci USA 83: 4028-4030 (1985); Creighton, T. E,, Proc. Natl. Acad. Sci, USA 83:5082-5086 (1988) Udgaonkar, J. B., et al., Nature 335:694-699 (1988) Roder, H., et al., Nature 335:700-704 (1988); and Mouschek, Nature 30:122-126 (1989) (all of these publications re incorporated herein by reference). Additionally, the role of disulfide bond formation has been investigated, see Maher, P. A., et al., Proc. Natl. Acad. Sci. USA 83:9001-9005 (1986); Goldenberg., D. P., et al., Nature 338:127-132 (1989); Matsumura, M., et al. Nature 342: 291-293 (1989) (all of these publications are incorporated herein by reference).
MHC molecules are the subject of Ju, U.S. Pat. No. 4,861,589; Sanderson, U.S. Pat. No. 4,478,823; Sanderson, U.S. Pat. No. 4,440,376; and Bach, et al., U.S. Pat. No. 4,265,873 (all of these patents are incorporated herein by reference).